Circulating endothelial progenitor cells (EPCs) derived from the bone marrow have been documented to contribute to tumor angiogenesis and ischemic limb revascularization, however, the contribution of marrow-derived cells to the pulmonary endothelium remains unclear. This proposal presents preliminary data showing that bone marrow-derived cells contribute to the pulmonary vascular endothelium, and that these cells arise from a pluripotent hematopoietic stem cell (HSC) with hemangioblastic potential. This observation suggests that enhancing this process could be adapted for the treatment of diseases involving the pulmonary vasculature by replacing damaged endothelial cells or by delivering therapeutic genes carried by engrafting cells. In order to develop a cell-based therapy for pulmonary vascular diseases, however, it is first crucial to understand: a) the precise role of bone marrow-derived cells in lung vascular repair; b) whether vascular reconstituting cells can be delivered directly after lung injury to rescue the injured host, and c) whether methods can be developed to augment endothelial engraftment. The specific aims of this grant proposal are designed to examine the roles of bone marrow HSCs in reconstituting the lung endothelium. Exposure to hyperoxia is employed to diffusely injure the lung endothelium of mice. During a two week recovery period after injury, the relative contributions of bone marrow-derived cells vs. native endothelial cells to the recovering endothelium is studied, Single cell transplantation is employed in order to confirm pluripotency and clonogenicity of the stem cells responsible for endothelial reconstitution, Finally, a cell-based therapy for pulmonary vascular diseases is developed through several approaches designed to augment the engraftment of bone marrow-derived cells in the injured lung endothelium. Over-expression of genes that promote self-renewing divisions of stem cells is used to expand HSCs in culture that can be infused into mice after lung injury. Finally, manipulation of cytokine signaling is used to mobilize and recruit bone marrow-derived cells for lung endothelial repair. Successful completion of the specific aims should help to advance research focused on cell-based therapies for lung diseases that involve injury or pathology of the pulmonary vasculature. PROJECT NARRATIVE. Injury to the blood vessels of the lung is responsible for considerable morbidity and mortality, and aberrant or incomplete repair of these vessels is characteristic of diseases such as pulmonary hypertension and acute respiratory distress syndrome. This proposal develops novel therapies, based on injections of hematopoietic stem cells, in order to rescue and repair the lining cells (endothelium) of these injured lung vessels. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]